Extensive surveys of the fatty acid composition of seed oils from different species of higher plants have resulted in the identification of more than 210 naturally occurring fatty acids which differ by the number and arrangement of double or triple bonds and various functional groups, such as hydroxyls, ketones, epoxys, cyclopentenyl or cyclopropyl groups, furans or halogens (van de Loo et al. 1993). At least 33 structurally distinct monohydroxylated plant fatty acids, and 12 different polyhydroxylated fatty acids have been described (reviewed by van de Loo et al. 1993; Smith, 1985).
The most commonly occurring fatty acids in both membrane and storage lipids are 16- and 18-carbon fatty acids which may have from zero to three, methylene-interrupted, unsaturations. These are synthesized from the fully saturated species as the result of a series of sequential desaturations which usually begin at the .DELTA.9 carbon and progress in the direction of the methyl carbon (Browse and Somerville, 1991). Fatty acids which cannot be described by this simple algorithm are generally considered "unusual" even though several, such as lauric (12:0), erucic (22:1) and ricinoleic acid (12D-hydroxyoctadec-cis-9-enoic acid) are of significant commercial importance. The biosynthesis of hydroxylated fatty acids such as ricinoleic acid in castor (Ricinus communis) seed is the subject of this invention.
The taxonomic relationships between plants having similar or identical kinds of unusual fatty acids have been examined (van de Loo et al., 1993). In some cases, particular fatty acids occur mostly or solely in related taxa. In other cases there does not appear to be a direct link between taxonomic relationships and the occurrence of unusual fatty acids. In this respect, ricinoleic acid has now been identified in 12 genera from 10 families (reviewed in van de Loo et al., 1993). Thus, it appears that the ability to synthesize hydroxylated fatty acids has evolved several times independently during the radiation of the angiosperms. This suggested to us that the enzymes which introduce hydroxyl groups into fatty acids arose by minor modifications of a related enzyme. Indeed, as noted below, this invention is based on our discovery that plant fatty acid hydroxylases are highly homologous to plant fatty acid desaturases.
A feature of hydroxylated or other unusual fatty acids is that they are generally confined to seed triacylglycerols, being largely excluded from the polar lipids by unknown mechanisms (Battey and Ohlrogge 1989; Prasad et al., 1987). This is particularly intriguing since diacylglycerol is a precursor of both triacylglycerol and polar lipid. With castor microsomes, there is some evidence that the pool of ricinoleoyl-containing polar lipid is minimized by a preference of diacylglycerol acyltransferase for ricinoleate-containing diacylglycerols (Bafor et al. 1991). Analyses of vegetative tissues have generated few reports of unusual fatty acids, other than those occurring in the cuticle. A small number of exceptions exist in which unusual fatty acids are found in tissues other than the seed.
Castor (Ricinus communis L.) is a minor oilseed crop. Approximately 50% of the seed weight is oil (triacylglycerol) in which 85-90% of total fatty acids are the hydroxylated fatty acid, ricinoleic acid (12D-hydroxyoctadec-cis-9-enoic acid). Oil pressed or extracted from castor seeds has many industrial uses based upon the properties endowed by the hydroxylated fatty acid. The most important uses are production of paints and varnishes, nylon-type synthetic polymers, resins, lubricants, and cosmetics (Atsmon 1989). In addition to oil, the castor seed contains the extremely toxic protein ricin, allergenic proteins, and the alkaloid ricinine. These constituents preclude the use of the untreated seed meal (following oil extraction) as a livestock feed, normally an important economic aspect of oilseed utilization. Furthermore, with the variable nature of castor plants and a lack of investment in breeding, castor has few favorable agronomic characteristics. For a combination of these reasons, castor is no longer grown in the United States and the development of an alternative domestic source of hydroxylated fatty acids would be attractive. The production of ricinoleic acid, the important constituent of castor oil, in an established oilseed crop through genetic engineering would be a particularly effective means of creating a domestic source.
The biosynthesis of ricinoleic (12D-hydroxyoctadec-cis-9-enoic) acid from oleic acid in the developing endosperm of castor (Ricinus communis) has been studied by a variety of methods. Morris (1967) established in double-labeling studies that hydroxylation occurs directly by hydroxyl substitution rather than via an unsaturated-, keto- or epoxy-intermediate. Hydroxylation using oleoyl-CoA as precursor can be demonstrated in crude preparations or microsomes, but activity in microsomes is unstable and variable, and isolation of the microsomes involved a considerable, or sometimes complete loss of activity (Galliard and Stumpf, 1966; Moreau and Stumpf, 1981). Oleic acid can replace oleoyl-CoA as a precursor, but only in the presence of CoA, Mg.sup.2+ and ATP (Galliard and Stumpf, 1966) indicating that activation to the acyl-CoA is necessary. However, no radioactivity could be detected in ricinoleoyl-CoA (Moreau and Stumpf, 1981). These and more recent observations (Bafor et al., 1991) have been interpreted as evidence that the substrate for the castor oleate hydroxylase is oleic acid esterified to phosphatidylcholine or another phospholipid.
The hydroxylase is sensitive to cyanide and azide, and dialysis against metal chelators reduces activity, which could be restored by addition of FeSO.sub.4, suggesting iron involvement in enzyme activity (Galliard and Stumpf, 1966). Ricinoleic acid synthesis requires molecular oxygen (Galliard and Stumpf, 1966; Moreau and Stumpf 1981) and requires AND(P)H to reduce cytochrome b5 which is thought to be the intermediate electron donor for the hydroxylase reaction (Smith et al., 1992). Carbon monoxide does not inhibit hydroxylation, indicating that a cytochrome P450 is not involved (Galliard and Stumpf, 1966; Moreau and Stumpf 1981). Data from a study of the substrate specificity of the hydroxylase show that all substrate parameters (i.e. chain length and double bond position with respect to both ends) are important; deviations in these parameters caused reduced activity relative to oleic acid (Howling et al., 1972). The position at which the hydroxyl was introduced, however, was determined by the position of the double bond, always being three carbons distal. Thus, the castor acyl hydroxylase enzyme can produce a family of different hydroxylated fatty acids depending on the availability of substrates. Thus, although we refer to the enzyme throughout as oleate hydroxylase it can more properly be considered an acyl hydroxylase of broad substrate specificity.
The only other organism in which ricinoleic acid biosynthesis has been investigated is the ergot fungus, Claviceps purpurea. Ricinoleate accumulates (up to 40% of the fatty acids) in the glycerides produced particularly by sclerotia of anaerobic cultures (Kren et al., 1985). As this suggests, oxygen is not necessary for the synthesis of ricinoleic acid in Claviceps, and the precursor of ricinoleic acid in fact appears to be linoleic acid (Morris et al., 1966). However, ricinoleic acid may not be formed simply by hydration of linoleic acid, since there are no free hydroxyl groups in ergot oil. Rather, the hydroxyl groups are all esterified to other, non-hydroxy fatty acids, leading to a range of tetra-acyl-, penta-acyl- and hexa-acyl-glycerides. These estolides may be formed by a direct enzymic addition of non-hydroxy fatty acids across the 12 double bond of linoleate (Morris, 1970). Ricinoleic acid may, therefore, be merely an artifact of the hydrolysis employed to study the fatty acid composition of the oil.
The castor oleate hydroxylase has many superficial similarities to the microsomal fatty acyl desaturases (Browse and Somerville, 1991). In particular, plants have a microsomal oleate desaturase active at the .DELTA.12 position. The substrate of this enzyme (Schmidt et al., 1993) and of the hydroxylase (Bafor et al., 1991) appears to be oleate esterified to the sn-2 position of phosphatidylcholine. The modification occurs at the same position (.DELTA.12) in the carbon chain, and requires the same cofactors, namely electrons from NADH via cytochrome b.sub.5 (Kearns et al., 1991; Smith et al., 1992) and molecular oxygen. Neither enzyme is inhibited by carbon monoxide (Moreau and Stumpf, 1981) the characteristic inhibitor of cytochrome P450 enzymes.
Conceptual basis of the invention
A feature of certain fatty acid modifying enzymes such as fatty acyl desaturases and castor oleate hydroxylase is that they catalyze reactions in which an unactivated C--H bond is cleaved. To catalyze this energetically demanding cleavage, these fatty acid modifying enzymes utilize the high oxidizing power of molecular oxygen. There are presently two known classes of enzyme cofactors capable of this type of O.sub.2 -dependent chemistry. The haem-containing oxygenases including cytochromes P450 are one class. However, as noted above, substantial evidence indicates that oleate hydroxylase is not a cytochrome P450 enzyme. The second class of cofactor known to be capable of this type of O.sub.2 -dependent chemistry is less well characterized, but is typified by the bacterial enzyme methane monooxygenase (van de Loo et al., 1993). The cofactor in the hydroxylase component of methane monooxygenase is termed a .mu.-oxo bridged diiron cluster (FeOFe). The two iron atoms of the FeOFe cluster are liganded by protein-derived nitrogen or oxygen atoms, and are tightly redox-coupled by the covalently-bridging oxygen atom. The catalytic cycle of methane monooxygenase is not so well understood as that of the P450 oxygenases, but there are known differences and similarities. Rather than two discrete single-electron reductions of the haem cofactor, the FeOFe cluster accepts two electrons, reducing it to the diferrous state, before oxygen binding. Upon oxygen binding, it is likely that heterolytic cleavage also occurs, leading to a high valent oxoiron reactive species that is very similar to that of the haem cofactor, but stabilized by resonance rearrangements possible within the tightly coupled FeOFe cluster, rather than through a porphyrin- or protein-derived ligand. The stabilized high-valent oxoiron state of methane monooxygenase is capable of proton extraction from methane, followed by oxygen transfer, giving methanol.
The FeOFe cofactor has been shown to be directly relevant to plant fatty acid modifications by the demonstration that castor stearoyl-ACP desaturase contains this type of cofactor (Fox et al., 1993). This desaturase is a member of a small family of plant fatty acid desaturases that are soluble enzymes, whereas most other desaturases are membrane-bound. Putative iron-binding motifs have been identified in the castor stearoyl-ACP desaturase primary structure by comparison to other soluble enzymes containing the FeOFe cluster (Fox et al., 1993). These similar motifs, (D/E)-E-X-R-H, are characteristically spaced approximately 90 residues apart in a number of soluble diiron-oxo proteins, including methane monooxygenase. Recently, cDNA clones for several plant membrane-bound desaturases encoding microsomal and plastid .omega.-3 and .omega.-6 desaturases of several plant species have been isolated (Arondel et al., 1992; Iba et al., 1993; Okuley et al., 1994; Yadav et al., 1993). Of great interest is the identification of a similarly repeated motif in all of these sequences (Schmidt et al., 1993), the membrane-bound rat stearoyl-CoA desaturase (Thiede et al., 1986) and in two membrane-bound monooxygenases (Kok et al., 1989; Suzuki et al., 1991). This motif, H-X-X-H-H in the desaturases and H-X-X-X-H-H in the monooxygenases, may be the functional equivalent in membrane-bound FeOFe proteins of the (D/E)-E-X-R-H motif in the soluble FeOFe proteins. This suggests that the plant membrane bound desaturases may also accomplish oxygen-dependent fatty acid desaturation through an FeOFe cofactor.
Of the well-characterized FeOFe-containing enzymes, methane monooxygenase catalyses a reaction involving oxygen-atom transfer (CH.sub.4 .fwdarw.CH.sub.3 OH), while the FeOFe cluster of ribonucleotide reductase catalyses the oxidation of tyrosine to form a tyrosyl cation radical without oxygen-atom transfer. However, site-directed mutagenesis of Phe208 to Tyr resulted in the conversion of this enzyme to an oxygen transfer catalyst, Tyr208 being hydroxylated and shown to be acting as a ligand to one iron of the FeOFe cluster. Therefore, the argument made for the P450 oxygenases catalyzing a range of reactions through the use of the same reactive intermediate modulated by the electronic and structural environment provided by the protein, can also be applied to FeOFe-containing enzymes. Modifications of the active site of plant fatty acid oxidizing enzymes containing FeOFe clusters could thus alter the outcome of the reaction, including whether oxygen-atom transfer occurs or not.
On the basis of the foregoing considerations, we hypothesized that the castor oleate hydroxylase is a structurally modified fatty acyl desaturase, based upon three arguments. The first argument involves the taxonomic distribution of plants containing ricinoleic acid. Ricinoleic acid has been found in 12 genera of 10 families of higher plants (reviewed in van de Loo et al., 1993). Thus, plants in which ricinoleic acid occurs are found throughout the plant kingdom, yet close relatives of these plants do not contain the unusual fatty acid. This pattern suggests that the ability to synthesize ricinoleic acid has arisen several times independently, and is therefore a quite recent divergence. In other words, the ability to synthesize ricinoleic acid has evolved rapidly, suggesting that a relatively minor genetic change was necessary to accomplish it. Several mechanisms for such facile evolution of a new enzyme activity are envisaged. One mechanism would be for the modification of a gene normally encoding a fatty acid hydroxylase active in the epidermis and involved in the synthesis of a hydroxy-fatty acid cutin monomer. The other mechanism would be for modification of a gene encoding a microsomal fatty acid desaturase, such that instead of performing one type of oxidation reaction (desaturation) it now performs another (hydroxylation).
The second argument is that many biochemical properties of castor oleate-12-hydroxylase are similar to those of the microsomal desaturases, as discussed above (eg., both preferentially act on fatty acids esterified to the sn-2 position of phosphatidylcholine, both use cytochrome b5 as an intermediate electron donor, both are inhibited by cyanide, both require molecular oxygen as a substrate, both are thought to be located in the endoplasmic reticulum).
The third argument stems from the discussion of oxygenase cofactors above, in which it is suggested that the plant membrane bound fatty acid desaturases may have a .mu.-oxo bridged diiron cluster-type cofactor, and that such cofactors are capable of catalyzing both fatty acid desaturations and hydroxylations, depending upon the electronic and structural properties of the protein active site.
Taking these three arguments together, it was hypothesized that oleate-12-hydroxylase of castor endosperm is homologous to the microsomal oleate .DELTA.12 desaturase found in all plants. When this invention was conceived, the structure of microsomal oleate .DELTA.12 desaturase (also known as .omega.-6 desaturase) was not known. However, based on the high degree of homology between plastid and endoplasmic-reticulum-localized .omega.-3 desaturases (Iba et al., 1993), we further hypothesized that the microsomal .DELTA.12 desaturase was homologous to the microsomal (.omega.-3) desaturase in particular, and also to the equivalent desaturases of the chloroplast inner envelope. A number of genes encoding microsomal .omega.-3 desaturases from various species have recently been cloned and substantial information about the structure of these enzymes is now known (Arondel et al., 1992; Iba et al., 1993; van de Loo and Somerville, 1993; Yadav et al., 1993). Hence in the following invention we teach how to use structural information about fatty acyl desaturases to isolate fatty acyl hydroxylase genes. Although, in the following example we reduce this invention to practice only for the castor oleate hydroxylase, this example unequivocally teaches the method by which any carbon-monoxide insensitive plant fatty acyl hydroxylase gene can be identified by one skilled in the art.
The invention is more fully described by reference to the following: